The objective of Project 3 is to characterize the role played by a highly novel form of chromatin regulation substitution of histone variants in the prefrontal cortex (PFC) and nucleus accumbens (NAc) in mediating depression- and antidepressant-like responses in animal models. Exciting new research, much of it from our laboratory, has demonstrated that, in addition to post-translational modifications of histones, eukaryotic cells, including mature neurons, further generate chromatin structural variation through the introduction of variant histone proteins into existing nucleosomes. We focus here on histone H3.3, which we have shown is enriched in mature neurons and dynamically regulated by neural activity. Its deposition depends in part on specific histone modifications, including those examined in Projects 1 and 2. Moreover, our data indicate that H3.3 is incorporated into nucleosomes at genes undergoing activation or repression in response to neuronal activity, neurotransmitters, and neurotrophic factors. Recently, we demonstrated that neural activation in vivo by use of optogenetic tools, or exposure to chronic stress, also regulates H3.3 expression in PFC and NAc. We have seen altered levels of H3.3 in the PFC and NAc of depressed humans as well. These highly novel findings set the stage for our proposed investigations. We will characterize regulation of H3.3 expression in PFC and NAc in both adult and early life stress models as well as map genome-wide stress-induced changes in the deposition of H3.3 in these models. Additionally, we will use biochemical methods, with which our laboratory has extensive expertise, to identify novel interacting proteins that associate with H3.3 in brain under basal and stress conditions. Finally, we will directly characterize the functional importance of H3.3 deposition in depression models by utilizing unique gain- and loss-of-function tools established by our group to investigate the effect of manipulating H3.3 within PFC or NAc neurons on responses to stress and antidepressant treatments at the behavioral, chromatin, and gene expression levels. Parallel studies of H3.3 will be carried out on PFC and NAc of depressed humans in conjunction with Project 4. These studies provide a template for the analysis of the roles played by other key histone variants in depression.